Use of an activation-specific probe to show that Rap1A and Rap1B display different sensitivities to activation by forskolin in Rat1 cells

Rap1A and Rap1B are small GTPases of the Pas superfamily whose activation c an be measured using a probe that interacts specifically with the GTP-bound forms of Rap1A and Rap1B. Using this procedure we demonstrate that the cyc lic AMP-elevating agent forskolin activates both Rap1A and Rap1B in Rat1 ce lls. Whilst the protein kinase A inhibitor H89 ablated the ability of forsk olin to cause cAMP response element binding protein (CREB) phosphorylation in Rat1 cells, it did not affect the ability of forskolin to activate eithe r Rap1A and Rap1B, Forskolin differentially activated Rap1A and Rap1B isofo rms in a time- and dose-dependent manner. The cAMP-specific type 4 family p hosphodiesterase inhibitor rolipram potentiated the rate of activation of b oth Rap1A and Rap1B by forskolin challenge of Rat1 cells. Challenge of Rat1 cells with rolipram alone was able to elicit the phosphorylation of CREB b ut not activation of either Rap1A or Rap1B. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reser ved.